Nucleic acid amplification reactions, such as polymerase chain reaction (PCR), are generally template-dependent reactions in which a desired nucleic acid sequence is amplified by treating separate complementary strands of a target nucleic acid with an excess of two oligonucleotide primers. The primers are extended to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence. In such processes, the nucleic acid sequence between the primers on the respective DNA strands is selectively amplified.
A variety of thermostable polymerases have been discovered that can be used in PCR. At least five families of DNA-dependent DNA polymerases are known, although most fall into families A, B and C. There is little or no structural or sequence similarity among the various families. Most family A polymerases are single chain proteins that can contain multiple enzymatic functions including polymerase, 3′ to 5′ exonuclease activity and 5′ to 3′ exonuclease activity. Family B polymerases typically have a single catalytic domain with polymerase and 3′ to 5′ exonuclease activity, as well as accessory factors. Family C polymerases are typically multi-subunit proteins with polymerization and 3′ to 5′ exonuclease activity.
Taq polymerase has inherent polymerase and 5′-3′ exonuclease activity, but does not have 3′-5′ exonuclease (“proofreading”) activity. Utilizing the inherent 5′ to 3′ exonuclease activity of Taq, it is possible to achieve PCR amplification and signal release from a target-specific fluorogenic probe (e.g., a “Taqman” probe). The 5′ to 3′ exonuclease activity of Taq cleaves the 5′ terminus of a hybridized oligo probe to release both mono- and oligonucleotides. The probe is hydrolyzed during strand replication so that the accumulating fluorescent signal correlates with amplification.
Pfu DNA polymerase and other family B polymerases has superior thermostability, inhibitor tolerance, and proofreading properties compared to Taq DNA polymerase. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3′ to 5′ exonuclease proofreading activity, meaning that it works its way along the DNA from the 5′ end to the 3′ end and corrects nucleotide-misincorporation errors. This means that Pfu DNA polymerase-generated PCR fragments will have fewer errors than Taq-generated PCR inserts. However, Pfu and other family B polymerases lack 5′-3′ exonuclease activity and thus do not work in probe-based quantitative PCR methods such as those involving Taqman probes.